Renal peritumoral adipose tissue undergoes a browning process and stimulates the expression of epithelial-mesenchymal transition markers in human renal cells

Tumor cells can interact with neighboring adipose cells and adipocyte dedifferentiation appears to be an important aspect of tumorigenesis. We evaluated the size of adipocytes in human adipose explants from normal (hRAN) and kidney cancer (hRAT); changes in the expression of WAT and BAT/beige markers in hRAN and hRAT; the expression of epithelial-mesenchymal transition (EMT) cell markers in human kidney tumor (786-O, ACHN and Caki-1); and non-tumor (HK-2) epithelial cell lines incubated with the conditioned media (CMs) of hRAN and hRAT. We observed that hRAT adipocytes showed a significantly minor size compared to hRAN adipocytes. Also, we observed that both Prdm16 and Tbx1 mRNA and the expression of UCP1, TBX1, PPARγ, PCG1α, c/EBPα LAP and c/EBPα LIP was significantly higher in hRAT than hRAN. Finally, we found an increase in vimentin and N-cadherin expression in HK-2 cells incubated for 24 h with hRAT-CMs compared to hRAN- and control-CMs. Furthermore, desmin and N-cadherin expression also increased significantly in 786-O when these cells were incubated with hRAT-CMs compared to the value observed with hRAN- and control-CMs. We observed a significant decrease in E-cadherin expression in the ACHN cell line incubated with hRAT-CMs versus hRAN- and control-CMs. However, we did not observe changes in E-cadherin expression in HK-2, 786-O or Caki-1. The results obtained, together with the results previously published by our group, allow us to conclude that perirenal white adipose tissue browning contributes to tumor development in kidney cancer. In addition, hRAT-CMs increases the expression of mesenchymal markers in renal epithelial cells, which could indicate a regulation of EMT due to this adipose tissue.


Results
The adipocytes that surround the kidney tumor are smaller than the adipocytes that surround a normal kidney. We evaluated the size of adipocytes from different renal adipose tissues. Specifically, we compared the hRAN and hRAT. We observed significant changes in the size of adipocytes in response to the presence of the tumor. The hRAT adipocytes showed a significantly smaller size compared to the hRAN adipocytes (Fig. 1). This change in adipocyte size, together with the increased expression of leptin and its receptor in hRAT versus hRAN 5 , suggest an increase of lipolysis by hRAT adipocytes compared to hRAN adipocytes.
hRAT showed an increase in gene expression of Prdm16 and TBX1 compared to hRAN. To evaluate browning of perirenal AT, we measured mRNA levels of Prdm16, TBX1, Ucp1 and PGC1 alpha in AT from normal and tumor kidney. Increased levels of Prdm16 and TBX1 mRNA in hRAT compared to hRAN (Fig. 2, p < 0.05) were found. No significant differences were found in Ucp1 and PGC1 alpha in hRAT versus hRAN mRNA. UCP1 and PGC1 alpha protein expression increased in hRAT adipocytes compared to hRAN. We performed immunohistochemistry assays on hRAN and hRAT to measure UCP1, PGC1 alpha and HSL protein levels and localization. UCP1 and PGC1 alpha protein abundance increased in hRAT adipocytes compared to hRAN adipocytes (Fig. 3, p < 0.05; negative controls are shown in Supplementary Fig. 1). Also, we observe the multilocular adipocyte morphology in hRAT compared to hRAN ( Supplementary Fig. 2).  Relative fold expression of Prdm16, TBX1, Ucp1 and PGC1 alpha gene expression from hRAN and hRAT. The mRNA profiles of Prdm16, TBX1, Ucp1 and PGC1 alpha from different adipose tissue were analyzed by qRT-PCR and normalized by their relative ratio to GAPDH. Data are mean ± SEM. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *p < 0.05. www.nature.com/scientificreports/ in HK-2 cells incubated for 24 h with hRAT-CMs compared to hRAN-and control-CMs (p < 0.05) (Fig. 5A,I).

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The same tendency was observed for the expression of desmin, but it was not statistically significant (Fig. 5E). Furthermore, desmin and N-cadherin expression also increased significantly in 786-O when these cells were incubated with hRAT-CMs compared to the value observed with hRAN-and control-CMs (Fig. 5F,J p < 0.05). However, no significant changes in vimentin expression were observed in 786-0 cells (Fig. 5B). Likewise, we did not find significant changes in the expression of vimentin, desmin or N-cadherin in the two tumor renal epithelial cell lines originating from metastatic sites (ACHN and Caki-1 cells) ( Fig. 5C-D,G-L). Furthermore, we evaluated changes in E-cadherin expression in the four cell lines incubated with hRAT-, hRAN-and control CMs. We observed a significant decrease in this epithelial marker in the ACHN cell line incubated with hRAT-CMs versus hRAN-and control-CMs (Fig. 5O). We did not observe changes in E-cadherin expression in any of the other cell lines tested (Fig. 5 M-P) ( Supplementary Fig. 4).

Discussion
Renal cancer (RCa) is considered to be the fifth most common type of cancer worldwide, having a high mortality rate in both men and women. Tumor development and maintenance of a cancerous phenotype, requires a bidirectional communication between epithelial cells and the stromal environment. Renal adipose tissue is one of the most abundant cell types surrounding renal epithelial cells 23 . Our group has shown that the periprostatic adipose tissue of patients with prostate cancer can regulate tumor behavior, both in early and late stages of the disease 24 . Furthermore, we have worked with adipose tissue fragments from human breast tumors (hATT) and normal breast glands (hATN), from which we obtained the corresponding CMs (hATT-CMs and hATN-CMs). We showed that hATT is capable of stimulating growth and metastatic capacity of mammary tumors by different mechanisms, unlike hATN 3,25,26 . Recently, we demonstrated that renal peritumoral adipose tissue undergoes a process of adaptation to changes locally generated by the tumor 5 . Also, we show that this hRAT is capable of stimulating protumorigenic behavior of renal epithelial cells 5 . We observed that hRAT expressed significantly higher amounts of leptin and ObR, relative to hRAN. It has been shown that leptin has a pro-tumorigenic function and can act by increasing lipolysis 14 . This increase in lipolysis is accompanied by a decrease in the size of adipocytes. Along these lines, our results show a significant decrease in the mean size of hRAT adipocytes with respect to the size of hRAN adipocytes (Fig. 1). The increased expression of both leptin and its receptor, together with the decrease in the size of hRAT adipocytes, allow us to suggest an increase in lipolysis in hRAT adipocytes. The increase in lipolysis would favor an increased availability of energy to the tumor, favoring its development. Leptin has been found to activate thermogenesis in BAT, increasing UCP1 production. It also exerts browning stimulation in WAT, with a greater number of mitochondria, and expressing UCP1 and other markers of browning, such as PRDM16 27 . Tumor induced differentiation to beige/brown adipose tissue is an important contribution to the hypermetabolic state of breast 21 and prostate 7 cancer, but to our knowledge, no previous research has been performed on browning and kidney cancer. However, Jespersen et al. 28 they showed that the perirenal fat in adult humans consist of dormant BAT, and small amounts of adipocytes with a multilocular morphology are present near regions with sympathetic activity. To begin to elucidate this, we evaluated the expression of different markers of WAT and BAT/beige adipocytes in hRAT and hRAN. The browning of WAT is triggered by increased gene expression levels of different markers involved in the BAT adipogenic differentiation, including PPAR gamma or PGC-1 alpha 28 . Furthermore, PPAR gamma induces the expression of C/EBP, which makes this gene a key regulator of WAT differentiation 29,30 . PPAR gamma also induces the expression and production of UCP-1 in these beige adipocytes 31 . UCP1 is a transmembrane protein that uncouples the electron transport chain (ETC) by pumping protons from the intermembrane space back into the mitochondrial matrix, thereby generating heat rather than ATP 32 . As well, PRDM16 is a master regulator gene of brown adipocyte differentiation and TBX1 beige adipocyte marker expression 18 . We observed that both Prdm16 and Tbx1 genes and protein expression of UCP1, TBX1, PPAR gamma, PCG1 alpha, c/EBPα LAP and c/EBPα LIP was significantly higher in hRAT than hRAN (Figs. 2, 3, 4). Considering our results together with those published by Jespersen et al. 28 , the increased browning found in hRAT compared to hRAN could be due to both stimulation of brown/beige adipogenesis of progenitors and/or transdifferentiation of white to brown/beige adipocytes. However, the human adipose explants from kidney samples used to perform the experiments were taken from regions far from sources of local sympathetic activity, that is, from remote areas in which reservoirs of a latent BAT state have been identified. This is the first work that demonstrates a transdifferentiation of WAT adipocytes in the human perirenal AT that surrounds a renal tumor, since we observed an increase in the amount of beige adipose tissue in hRAT, as opposed to hRAN. In light of these results, together with those previously described 5 , we postulate that the renal tumor would be regulating the differentiation of the surrounding WAT and its browning. The browning process might have a role in the development of kidney tumors. Currently, and to deepen our understanding of this possible regulation, we set out to study changes in WAT and BAT/beige adipocytes markers in hRAN fragments incubated with MCs from tumor kidney cell lines. Preliminary results (not yet published) allow us to observe an increase in the expression of BAT/beige adipocyte markers in hRAN, which would support the hypothesis that the renal tumor would be stimulating browning of the surrounding AT. On the other hand, we did not observe changes in the expression of E-cadherin in our experimental condition. This result could be due to partial and not total EMT, which has already been described in cancer 36 . Likewise, it has been seen that leptin is capable of stimulating EMT. Previously we showed that hRAT expressed significantly higher amounts of leptin and ObR, relative to hRAN 5 . It is known that leptin induces a fibroblastoid morphology evidenced by the decrease in the expression of epithelial markers (occludin, E-cadherin) and an increase in mesenchymal markers (fibronectin, N-cadherin, and vimentin) in breast cancer 37 . Therefore, we postulate that this adipokine could be involved in increasing the expression of EMT markers. Future experiments should use leptin neutralization antibodies in the conditioned media, to confirm our hypothesis about the importance of this adipokine in the observed effects. This is the first report demonstrating that hRAT presents beige/brown adipocyte characteristics, unlike hRAN. This browning process could be stimulated by the kidney tumor itself, and play an important role in renal tumor development. Furthermore, we demonstrate that hRAT produces soluble factors that facilitate the acquisition of a mesenchymal phenotype in cells that have not yet migrated. Sample collection and handling. Patients with suspected kidney cancer or healthy kidney donors were enrolled. After signing the informed consent, subjects were interviewed using a standard questionnaire that requested information about socio-demographic, medical, and lifestyle factors. Human adipose tissue explants from cancerous (hRAT; n = 23) kidneys were obtained from patients to whom a partial or total (tumor) nephrectomy was performed. Human adipose tissue explants from normal kidneys (hRAN; n = 19), were obtained from live kidney donors who had not received previous chemotherapy or radiotherapy treatment. Perirenal adipose tissue biopsies were taken as follows: (1) in the case of living kidney donors, the adipose tissue fragment was taken 1 cm away from the kidney. From the middle zone (middle pole); (2) in the case of patients with renal tumor, the fragment of adipose tissue was taken 1 cm from the kidney, also taking into account the location of the tumor. Trying, in all cases, to take the sample 1 cm from the location of the tumor, getting as close as possible to the middle zone. In all cases, biopsies were taken distant from the adrenal gland (sources of norepinephrine).

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The median body mass index (BMI) of patients was: 26.8 kg/m 2 for patients with renal tumor (hRAT), and 24.9 kg/m 2 for living kidney donors (hRAN). BMI (kg/m2) was calculated as weight (kg) divided by height (m) squared.
Samples were transported in PBS and processed immediately. On average, 2 h elapsed from the acquisition of the surgical sample until it was processed under a sterile laminar flow hood. The project was approved by the Medical School's ethics committee (Universidad Nacional de Cuyo, Argentina) according to the Declaration of Helsinki of experimentation with human subjects. All patients gave their informed consent to undergo tissue harvesting for this research 5 .
Gene expression by RT-qPCR analysis. Total RNA was extracted from 100 mg of tissue using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and quantified according to its absorbance at 260 nm (NanoDrop 2000, Thermo Scientific, Wilmington, USA). Contaminating genomic DNA was degraded with DNAse RQ1 (Promega, Madison, USA), cDNA was synthesized from one microgram of total RNA using 300 pmol oligo-dT primers, 10 mM dNTP (Thermo Scientific, Wilmington, USA) and 200U M-MLV reverse transcriptase (Promega, Madison, USA). Real-time qPCR was performed in a final volume of 20 uL containing 50 ng cDNA, 3 mM MgCl2, PCR LightCycler-DNA Master SYBRGreen reaction mix (Roche, Indianapolis, USA) and 0.5 mM of each specific primers (Table 1). Amplification was performed using a LightCycler thermocycler (Roche, Indi- Table 1. primer pair sequence are shown for the Forward (F) and Reverse (R) primers used to measure mRNA abundance by RT-qPCR.
H&E staining. Tissues (hRAT and hRAN) were fixed in 4% formaldehyde and embedded in paraffin. They were afterwards cut into sections of 5 μm thickness with a microtome, deparaffinized and stained with hematoxylin-eosin (H&E). Images were taken with a Nikon Eclipse E200 Microscope fitted with a digital still camera Micrometric SE Premium (Nikon Corp., Japan) at 100× magnification. Adipocyte area quantification (measuring adipocyte perimeter) in the three tissue types was performed in 8-10 fields of each preparation as mentioned above 5 .
Immunohistochemistry. 10 µm serial cuts were performed on the same tissue samples embedded in paraffin used for H&E staining. UCP1, PGC1 alpha and HSL expression were studied by means of immunohistochemistry. Briefly, hRAN and hRAT microtome slides were first deparaffinized, and then a heat-mediated antigen retrieval, endogenous peroxidase blocking and nonspecific tissue blocking were performed. Slides were then incubated with the different primary antibodies (Anti-UCP-1. SIGMA U6382. Dilution of 1:500; Anti-PGC1 alpha. Abcam ab54481. Dilution of 1:300; and Anti-HSL. Abcam ab45422. Dilution of 1:300) at 4 °C. And after that with an anti-rabbit biotinylated secondary IgG antibody. Finally, slides were incubated with peroxidaseconjugated streptavidin. Peroxidase reaction was performed with chromogen 3,3′-diaminobenzidine (DAB) (DAKO LSAB + Kit, HRP). Hematoxylin counterstaining was performed. Serial cuts incubated in the absence of the primary antibody were used as negative controls. Images were taken with a Nikon Eclipse E200 Microscope fitted with a Micrometric SE Premium (Nikon Corp., Japan) digital still camera at 10× and 40× magnification. DAB staining quantification in the three tissue types was performed in 5 fields of each preparation as mentioned above [3][4][5] .

Preparation of conditioned media (CMs) from hRAN and hRAT .
Adipose tissues were washed with cold PBS 1X (Gibco, USA and weighed. hRAN or hRAT were plated in culture flasks with M199 culture medium (Invitrogen™; 1 g tissue/10 ml M199), and incubated for 1 h at 37 °C in 5% CO 2 . After that, the medium was removed and replaced with fresh medium and the tissues were incubated for 24 h. Subsequently, the supernatant was collected and filtered using filters with 0.22 µm membranes. Then, supernatants were aliquoted into 1 ml fractions and immediately stored at − 80 °C. The control-CMs were obtained from the collection of the serumfree M199 medium after 24 h of incubation in a culture flask at 37 °C in 5% CO Western blot analysis. In order to evaluate protein expression levels, Western blots were performed.
UCP1, TBX1, PPARγ, PCG1 alpha, c/EBPα LAP, c/EBPα LIP, adiponectin, leptin, vimentin, desmin, N-cadherin and E-cadherin were measured after incubation of the epithelial cell lines with the different CMs obtained. The cells were lysed with Ripa buffer. Total protein in samples was quantified using the Pierce BCA protein assay kit (Thermo Scientific). Proteins were separated in a SDS-PAGE 12 gel, and electrotransferred to a PVDF membrane (Bio-Rad, USA). The membrane was later blocked with human serum albumin (Sigma-Aldrich, 0055 K) and then incubated with the different antibodies ON at 4 °C. The membranes were later washed, and incubated with proper secondary antibodies conjugated with biotione, and subsequently the signal was amplified with streptavidin. Antibody complexes were visualized by means of chemiluminescence (ECL; GE Helathcare). Membrane exposed images were obtained with the Chemidoc MP system (Bio-Rad, USA) and bands were quantified by www.nature.com/scientificreports/